The purpose of this research project is to study the mechanisms and regulation of the enzymes pyridoxal kinase and pyridoxine-5-p oxidase involved in the metabolism of vitamin B6 in brain tissues. The stereochemical course of pyridoxal kinase catalysed phosphoryl transfer will be examined using ATP, possessing a chiral Alpha-phosphorus, as a substrate. After analysis of the configuration of the product of the reaction, i.e., pyridoxal-5-p, one should be able to establish whether or not the phosphoryl group has been transferred directly between the two bound substrates. The topography of the catalytic site of the kinase will be studied using pyridoxyl derivatives of ATP and ADP, which are able to recognize the binding sites of the kinase and behave as competitive inhibitors with respect to ATP and pyridoxal. The bifunctional inhibitors display fluorescence properties that can be exploited to determine their mode of binding to the catalytic domains of the kinase. The regulation of the catalytic activity of pyridoxine-5-p oxidase by the product pyridoxal-5-p will be examined in detail. Experiments are designed to demonstrate that pyridoxal-5-p binds to a critical lysyl residue positioned in the catalytic domain of the oxidase. Pyridoxal kinase and pyridoxine-5-p oxidase interact in solution forming "clusters" of large molecular weight. The dissociation constant of such "enzymatic clusters" has been estimated to be lower than luM. The stability and stiochiometry of binding of the two cytosolic enzymes will be examined by a combination of hydrodynamic techniques, analytical ultracentrifugation and time resolved emission anisotropy. Affinity chromatography techniques using antibodies immobilized on protein-a sepharose will be used to detect the effects of the effectors pyridoxine-5-p and pyridoxal-5-p on the stability of the "enzyme clusters."